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Int. J. Adv. Res. Biol. Sci. (2017). 4(6): 72-78

International Journal of Advanced Research in Biological Sciences ISSN: 2348-8069

www.ijarbs.com DOI: 10.22192/ijarbs

Coden: IJARQG(USA)

Volume 4, Issue 6 - 2017

Research Article DOI: http://dx.doi.org/10.22192/ijarbs.2017.04.06.012

Isolation and identification of Escherichia coli and Salmonella sp. from apparently healthy Turkey 13

Jotan Kar1 , Tapas Roy Barman2, Arup Sen3, Sabuj Kanti Nath4*

Department of Microbiology and Veterinary Public Health, Chittagong Veterinary and Animal Sciences University (CVASU), Chittagong, Khulshi-4225, Bangladesh 2 Department of Medicine and Surgery, Chittagong Veterinary and Animal Sciences University (CVASU), Chittagong, Khulshi-4225, Bangladesh 4 Department of Animal Science and Animal Nutrition, Chittagong Veterinary and Animal Sciences University (CVASU), Chittagong, Khulshi-4225, Bangladesh. *Corresponding author: [email protected]

Abstract A study was designed with the aim of isolation and identification of Escherichia coli and Salmonella sp. in order to identify the prevalence of these bacteria in a turkey flock. For this, 120 (n) cloacal swab samples were collected from the apparently healthy turkey of a farm at Foujdarhat Cadet College Campus, Chittagong. The study was performed in between April and May 2016 where 120 swab samples were tested by using standard laboratory protocol in Poultry Research and Training Centre, Chittagong. Among biochemical tests, Indole test was done for the confirmation of E. coli where TSI agar slant was done for ensuring the positive Salmonella sp. Here, 90 (75%) samples were found positive for E. coli and 42 (35%) were for Salmonella sp. However, 21 (17.5%) samples had mixed infection with both E. coli and Salmonella sp. In this study, the prevalence of Salmonella infection according to sex and laying, the non-laying category the result was found statistically significant (p ≤ 0.05); similarly, in case of E. coli the prevalence between flock-1 and flock-2 also showed significant (p ≤ 0.05) findings. It was observed that Turkey generally acts as a carrier of a vast range of organisms including E. coli and Salmonella sp. This is first time studied to investigate E. coli and Salmonella sp. from Turkey in Chittagong, Bangladesh. So, further study is highly recommended.

Keywords: Prevalence, diarrhea, Escherichia coli, Salmonella spp.

Introduction 1996 and Kamal et al., 1988). Thereby those are responsible for mortality, reduced egg production, and hatchability as well (Barnes et al., 1997). The intestinal tract of poultry consists of complex and dynamic microbial community comprising primarily of bacteria (Zhao et al., 2001). The large family Enterobacteriaceae includes Gram (-ve) bacteria along with many harmful symbiotics such as Salmonella spp., E. coli, Yersinia pestis, Klebsiella, and Shigella.

In Bangladesh, poultry production system is grouped into the traditional and commercial system (Haque et al., 1991). Commercial rearing system of poultry for meat and egg has created a new era in the present time. Poultry, namely domestic chickens, turkeys, geese, and guinea fowls are kept throughout the world. Infections with bacteria of the genus Salmonella sp. and E. coli bring about a variety of acute and chronic diseases of poultry in Bangladesh (Bhattacharjee et al., 72


Other disease-causing bacteria in this family include Proteus, Enterobacter, Serratia, and Citrobacter (Garrity et al., 2004). Escherichia coli is a Gram (-ve), rod-shaped, facultatively anaerobic bacteria. This pathogenic E. coli can be categorized based on serogroups, pathogenic mechanisms, variation in epidemiology and different interaction with the intestinal mucosa, clinical symptoms or virulence factors (Kaper, 2005). Mainly they are-(1). Enterotoxigenic E. coli, (2). Enteroinvasive E. coli, (3). Enteropathogenic E. coli, (4). Enterohemorrhagic E. coli and (5). Enteroaggregative E. coli. Colibacillosis refers any localized or systemic infection caused entirely or partly by Avian Pathogenic Escherichia coli (APEC) including coli septicemia, coli granuloma, cellulitis, swollen head syndrome, peritonitis, salpingitis, osteomyelitis, synovitis, panophthalmitis, and omphalitis (Barnes et al., 1997). It is one of the principal causes of morbidity and mortality in chickens and turkeys (Blanco et al., 1998). The majority of the economic loss results from the mortality of the affected bird (Otaki, 1995). Environmental contamination, poor ventilation, contaminated water; improper hygiene practices favor the E. coli infection (Samad, 2005). Infection in young chicken and omphalitis may due to the entry of organisms through penetration prior to incubation of eggs (Weinak et al., 1981).

infection has occurred many of the birds excrete Salmonella organisms and contaminate the environment (Poppe, 1996)..

Materials and Methods During UVH placement, one turkey flock owner, from nearby Foujdarhat Cadet College Campus, Salimpur, Chittagong; came to our reputed SAQTVH hospital unit for getting treatment of three turkeys having severe diarrhea. He also mentioned that he had total 24 turkeys affected with diarrhea among total 120 turkeys in the flock. In this regard, considering the above facts, the present study was undertaken to find out actual causal agent of such types of illness to the birds, and thereby, the prevalence of underlying pathogen. Study area and study duration The study was conducted from April to June 2016 on 120 turkey samples at the nearby Foujdarhat Cadet College Campus, Salimpur, Chittagong. Sample collection The samples were collected from cloacae of turkey through pre-sterilized cotton swab and immediately transferred into screw capped test tubes containing buffered peptone water. Thermo flask containing ice was used to transport the samples from the collection site to Poultry Research and Training Centre (PRTC) Laboratory for analysis. Collected samples were preserved in a refrigerator at 4ºC until screening out the bacteria.

Poultry is the natural hosts for both of this organisms Salmonella pullorum and Salmonella gallinarum (Snoeyenbos et al., 1991). Salmonellosis is the causative agent of pullorum disease, fowl typhoid and fowl paratyphoid (Gomis et al., 1997). Pullorum disease is usually confined to the first 2-3 weeks of bird age and occasionally occurs in adults (Shivaprashad et al., 1997). Fowl typhoid is an acute and chronic disease which is caused by Salmonella gallinarum and causes a serious problem such as mortality, lowered egg production, and reduced hatchability by affecting the mature birds (Christensen et al., 1997). The vertical transmission has occurred via infected eggs and the horizontal through contaminated utensils and others infected materials (Shivaprashad et al., 1997 and Snoeyenbos et al., 1991). The incidence of salmonellosis in poultry has been well established in many countries eg. United States, Belgium, U. K., Malaysia, Spain and Japan; and the level of contamination by Salmonella ranged between 20-89% from total poultry population (Capita et al; 2003). The Center for disease control and prevention estimates that Salmonella in food is responsible for 1.3 million illness; 15000 hospitalizations and over 500 deaths a year. Once the

Data collection All the data including flock size, flock number, sex, age, duration of illness etc. of the individual birds were noted in a structured record keeping questionnaire. Media used for isolation Peptone water (Oxoid Ltd; pH 6.2±0.0) was used as primary enrichment media for E. coli and Salmonella sp. 4 selective media were used for the isolation of these bacteria. The Mac-Conkey agar (Oxoid Ltd, pH 7.4±0.2), EMB agar (Merck, pH 7.1±0.2) were used for E. coli where XLD agar (Oxoid Ltd, pH 7.4±0.2), and SS agar (Merck, pH 6.9±0.2) were used for Salmonella sp.

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Culture procedures for both E. coli and salmonella sp

blackish growth in SS agar were considered as presumptive Salmonella positive.

1ml of swab suspension was inoculated in a screw cap test tube containing 10ml of nutrient broth and incubated at 37ºC for 24 hours. Then for E. coli, samples were streaked on Mac-Conkey agar and incubated overnight. The growth of E. coli produces large, pink colonies on an agar plate. Subculture was then performed on EMB agar at 37ºC for 24 hours. The characteristic metallic sheen colonies were suggestive for E. coli positive. Then for further confirmation, biochemical test (Indole test) was done. For Salmonella sp., after inoculation of the sample on nutrient broth, one loop-full of the colony from broth was streaked on XLD agar plate and incubated at 37ºC for 24 hours. Then the positive samples were further inoculated on SS agar and incubated overnight at 37ºC for 24 hours. After incubation, colonies were observed. The colony with a black center in XLD and

Biochemical tests Tests for Escherichia coli E. coli produces strong acids and usually gas forms by the fermentation of d-glucose (positive in the methyl red test) and do not produce acetyl-methyl carbinols (acetoin) (negative in the Voges-Proskauer test). Lysine is decarboxylated by the majority of the strains. Indole test is also positive in case of E. coli. Indole test

Indole test was performed to determine the ability of an organism to form indole compound by splitting of amino-acid tryptophan. The positive result was noted by pink colored ring formation after addition of reagent. Table1: Different biochemical tests for E. coli

Test result Positive Negative

Indole test Pink ring No ring

Biochemical tests for Salmonella spp.

stabbing the butt down to the bottom and then streaked over the surface of the slant. Then, the TSI slant was incubated overnight at 37ºC.

TSI slant for identification of Salmonella spp A straight inoculating needle was used to take isolated colony from culture. The TSI slant was inoculated by

Table2: Biochemical test (TSI agar slant) for identification of Salmonella sp Test result Salmonella positive

TSI slant (Triple Sugar Iron test) Production of H2 S; blackening of the medium.

Preservation of culture

Results

All the positive isolates were inoculated into Trypticase Soy Broth (TSB) (Oxoid, England), incubated overnight at 37ºC and then stored at -80ºC adding 15% glycerol in 1.5 ml Eppendorf tubes for further research.

In this study, 120 cloacal swab samples were studied where 90 samples were positive for E. coli and given a prevalence of 75%. The positive samples were confirmed E. coli by Indole test which showed 100% sensitivity. In case of Salmonella sp., from 120 cloacal swab samples, it was observed that 42 samples were positive which given a prevalence of 35%. Biochemical test of positive samples of Salmonella sp. was performed by TSI agar slant that also showed 100% sensitivity.

Data analysis Obtained data was imported to the Microsoft Office Excell-2007 and transferred to the software STATA/IC-11 for analysis. Descriptive analysis was done. An association was regarded as significant if the p value was ≤0.05. 74


Pink color colony with black centre

Fig. 1: Salmonella colonies on XLD agar

Single black colony of Salmonella spp.

Fig. 2: Salmonella colonies on S-S agar

Production of H2 S; blackening of the medium Fig. 3: Salmonella on TSI agar slant

Disinctive metalic green sheen colony

Fig. 3: E. coli in EMB agar

Pink color colony

Fig. 3: E. coli in MacConkey agar

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Table3: The prevalence of E. coli and Salmonella sp. according to various traits Trait

Identification for E. coli

Sex Salmonella spp.

Variables

Negative

Positive

Total

Female Male Total Female Male Total

18 (60%) 12 (40%) 30 24 (30.77%) 54 (69.23%) 78

33 (36.7%) 57 (63.3%) 90 27 (64.3%) 15 (35.7%) 42

51(42.5%) 69 (57.5%) 120 51 (42.5%) 69 (57.5%) 120

3-5 6 (20%) 54 (60%) 60 (50%) ≤ 2.5 15 (50%) 15 (16.7%) 30 (25%) ≥ 6.5 9 (30%) 21 (23.3%) 30 (25%) Age Total 30 90 120 (m) 3-5 51 (65.38%) 9 (21.43%) 60(50%) Salmonella spp. ≤ 2.5 15(19.23%) 15 (35.7%) 30(25%) ≥ 6.5 12 (15.38%) 18 (42.7%) 30(25%) Total 78 42 120 Laying 9(30%) 21 (23.33%) 30(25%) E. coli Non 21(70%) 69(76.7%) 90(75%) Layer/ laying NonTotal 30 90 120 layer Laying 12 (15.4%) 18(42.9%) 30(25%) Salmonella spp. Non 66(84.6%) 24(57.1%) 90(75%) laying Total 78 42 120 Flock-1 15(50%) 75 (83.3%) 90 (75%) E. coli Flock-2 15(50%) 15(16.7%) 30(25%) Flock Total 30 90 120 Flock-1 63(80.7%) 27(64.3%) 90(75%) Salmonella spp. Flock-2 15(19.23%) 15(35.7%) 30(25%) Total 78 42 120 *Flock-1 (3-5 month and ≥ 6.5 month aged) and Flock-2 (≤ 2.5 month aged). E. coli

Here, according to male and female Turkey their present no significant (p>0.05) variation in case of E. coli. But, in the case of Salmonella sp. there had a significant (p ≤ 0.05) variation between male and female turkey. In this study, it was observed that 60 % turkey, aged 3-5 months were positive for E. coli whereas, 42.7% turkey, aged ≥6.5 months were positive for Salmonella sp. Though; there have no significant variation among the aged birds. This is interesting that 76.7 % non-laying birds were positive for E. coli and 42.9% laying birds were positive for Salmonella sp. There were significant variations between laying and non-laying birds in case of Salmonella sp. Although, the prevalence of E. coli between flock-1 and flock-2 showed statistically significant variation (p ≤ 0.05) where 83.3% turkey of flock-1 were positive, but in case of Salmonella sp.

p value 0.196

0.041

0.615

0.83

0.673

0.05

0.04

0.251

either flock-1 or flock-2 there had no significant findings.

Discussion The aim of this study was to focus on isolation and identification of the E. coli and Salmonella sp. from cloacal swabs and find out the prevalence of these infections on a turkey flock affected with severe diarrhea. Earlier, Cuiwei Zhao et al., 2001 found 11.9% E. coli infection in a commercial turkey flock where, Mohammad Jahantigh et al., 2015 found 14.8% Salmonella sp. The CSPI (Center for Science in the Public Interest) under Washington-US Department of Agriculture (USDA) showed that 13% of turkeys were contaminated with Salmonella sp., which was slightly lower than this study. However, in the study, overall prevalence of E. coli and Salmonella sp. were 75% and 35% respectively and prevalence of E. coli 76


were comparatively very much higher than the prevalence of Salmonella sp. In an experimental study of Narayan Rath et al., 1999 and G. R. Huff et al., 2015 observed comparatively higher mortalities and air-sacculitis in males (significant with p≤0.05) than females in a 5-week old turkey flock. Here, according to male and female Turkey there present no significant (p>0.05) variation in case of E. coli prevalence but, in case of Salmonella sp. there had a significant (p≤0.05) variation between male and female turkey with higher prevalence in females. In a previous study by the European Union (EU) on 2006-2007, showed 30.7% Salmonella-positive on non-laying 4-5 months aged turkeys but in this study it was observed as 57.1% which have a significant variation (p ≤0.05). According to EU, in layer turkey aged ≥6.5 months, Salmonella prevalence was 13.6% which is found 42.7% in the study. Islam et al., 2006 reported as the prevalence of Salmonella infection increased with the increase of age which also similar to my findings. Between the two flocks (Flock-1 and Flock-2) there had a significant (p ≤0.05) variation in E. coli infection. In the case of Salmonella sp., Hossain et al., 2010 found 34.2% Salmonella infection in large flocks (≥5001 birds) in comparison to 21.3% in the small (≤1000 birds) flocks. It is quite similar to the study. It might be due to a study on the non-commercial and experimental farm having smaller birds population where 17 female birds among the total of 40 birds, geographical variation and strains of the organisms.

Bhattacharjee, P.S., Kundu, R.L., Mazumder, J.U., Hossain, E. and Miah A.H. 1996. A retrospective analysis of chicken diseases diagnosed at the Central Disease Investigation Laboratory, Dhaka, Bangladesh. Bangladesh Veterinary Journal, 30: 105-113. Blanco, J.E., Blanco, M., Mora, A., Jansen, W.H., Garcı́a. V., Vázquez, M.L. and Blanco, J. 1998. Serotypes of Escherichia coli isolated from septicaemic chickens in Galicia (northwest Spain). Veterinary microbiology, 61 (3): 229-235. Capita, R., Alvarez, A.M., Alonso, C.C., Moreno, B. and Garcia, F.D.C. 2003. Occurrence of Salmonella in retail chicken carcasses and their products in Spain. International journal of food microbiology, 81 (2): 169-173. Center for Science in the Public Interest (CSPI); (http: //cspinet. org/) and Washington-United States Department of Agriculture (USDA); (http: //www. usda. gov/). Christensen, J.P., Brown, D.J., Madsen, M., Olsen, J.E. and Bisgaard, M. 1997. Hatchery borne Salmonella enteric serovar tennessee infections in broilers. Avian Pathology, 26: 155-167. Garrity, G.M,, Bell, J.A. and Lilburn, T.G. 2004. Taxonomic outline of the prokaryotes. Bergey's manual of systematic bacteriology. Springer, New York, Berlin, Heidelberg. p: 236-238. Gomis, S.M., Goodhope, R., Kumor, L., Caddy, N., Riddell, C., Petter, A.A. and Allan, J.J. 1997. Experimental reproduction of Escherichia coli, cellulitis and septicemia in broiler chickens. Avian diseases, 41: 234-240. Haque, M.E., Hamid, M.A., Howleder, M.A.R. and Haque, Q.M.E. 1991. Performance of native chicks and hens reared together or separately under rural condition in Bangladesh. Bangladesh Veterinary journal, 8: 11-13. Hossain, K.M.M., Hossain, M.T. and Yamato, I. 2010. Seroprevalence of Salmonella and Mycoplasma gallisepticum infection in chickens in Rajshahi and surrounding districts of Bangladesh. International Journal of Biology, 2 (2): 74. Huff, G.R., Huff, W.E. and Rath, N.C. 2015. Effect of dexamethasone on bacteriostatic activity of turkey monocytes and implications for food safety. Veterinary immunology and immunopathology, 166 (3): 151-158. Islam, M.M., Haider, M.G., Chowdhury, E.H., Kamruzzaman, M. and Hossain, M.M. 2006. Seroprevalence and pathological study of Salmonella infections in layer chickens and isolation and identification of causal agents.

Conclusion The current study revealed the presence of E. coli and Salmonella sp. in the cloacal swab of a turkey flock. The prevalence of E. coli and Salmonella sp. in the flock were 75% and 35% respectively. The E. coli infection was simply higher than the Salmonella infection. The high prevalence of Salmonella and E. coli in Turkey could be a threat to public health. Hence, it is an alarming issue for turkey farms. Finally, the comprehensive planned study should be needed to characterize the various serotypes of E. coli and Salmonella sp. and identify the prevalence pattern with antimicrobial resistance to tackling this alarming point.

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Quick Response Code DOI:10.22192/ijarbs.2017.04.06.012

How to cite this article: Jotan Kar , Tapas Roy Barman, Arup Sen, Sabuj Kanti Nath. (2017). Isolation and identification of Escherichia coli and Salmonella sp. from apparently healthy Turkey. Int. J. Adv. Res. Biol. Sci. 4(6): 7278.

DOI: http://dx.doi.org/10.22192/ijarbs.2017.04.06.012

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